The invention relates to a method for producing vectors containing a gene coding for an enzyme having reduced or deactivated feedback inhibition and to the use thereof for producing amino acids and nucleotides
Amino acids are used in human medicine, in the pharmaceutical industry, the food industry, and particularly in animal nutrition. Amino acids are known to be produced by the fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum, and of enterobacteria, in particular Escherichia coli. Due to their great significance, efforts are continually underway to improve production methods. Improvements to the process can relate to measures concerning fermentation techniques, such as stirring and the supply of oxygen, or to the composition of the nutrient media, such as the sugar concentration during fermentation or processing into product form by ion exchange chromatography, for example, or to the intrinsic performance characteristics of the microorganism itself.
Mutagenesis, selection, and mutant selection methods are employed to improve the performance characteristics of these microorganisms. In this way, strains are obtained that are resistant to antimetabolites or are auxotrophic for regulatorily significant metabolites and produce amino acids. Likewise, recombinant DNA methods are used to improve strains of L-amino acid producing strains by amplifying individual amino acid biosynthesis genes and analyzing the effect on amino acid production.
The synthesis of amino acids in microorganism takes place by way of synthesis pathways, which are referred to as amino acid biosynthesis pathways, amino acid synthesis pathways, or synthesis pathways. These synthesis pathways are composed of individual steps, in which the amino acid is synthesized from precursors. These precursors are provided in the central metabolism. They include, for example, pyruvate, alpha-ketoglutarate, oxaloacetate, pentose phosphate, acetyl-CoA, erythrose phosphate, phosphoenolpyruvate or phosphoglycerate. Moreover NADPH2, NH4+ and reduced tetrahydrofolate are required to synthesize amino acids from precursors using the synthesis pathways. The synthesis of amino acids from precursors using synthesis pathways in coryneform bacteria and enterobacteria is known to those skilled in the art. This relates to the amino acids L-alanine, L-valine, L-leucine, L-asparagine, L-aspartate, L-lysine, L-methionine, L-threonine, L-isoleucine, L-histidine, L-glutamate, L-glutamine, proline, glycine, L-arginine, L-tryptophan, L-tyrosine, L-phenylalanine, L-serine and L-cysteine. The synthesis of amino acids has been studied particularly well in Escherichia coli and Corynebacterium glutamicum. 
The synthesis pathways consist of a series of reactions catalyzed by enzymes and are subject to strict regulations. Particularly strict regulation is achieved by feedback-resistant enzymes. In wild type strains, strict regulating mechanisms prevent the production of metabolic products, such as amino acids, beyond the subsistence level needed and the delivery thereof to the medium. From a manufacturer's view, the construction of strains overproducing organic-chemical compounds therefore entails the need to overcome these metabolic regulations. These feedback-resistant enzymes preferably catalyze input reactions of the synthesis pathways or branch points on the synthesis pathways. The regulation often takes place via the end product of the synthesis pathway, which is to say the amino acids L-alanine, L-valine, L-leucine, L-asparagine, L-aspartate, L-lysine, L-methionine, L-threonine, L-isoleucine, L-histidine, L-glutamate, L-glutamine, proline, glycine, L-arginine, L-tryptophan, L-tyrosine, L-phenylalanine, L-serine and L-cysteine.
Intermediates can also intervene in a regulating manner, such as O-acetylserine, O-acetylhomoserine, O-succinylserine, O-succinylhomoserine or adenosylmethionine. Regulation is such that, at an increased concentration of the listed amino acids or intermediates, the respective enzyme of the synthesis pathway, or the feedback-resistant enzymes of the synthesis pathway, are inhibited.
Examples of enzymes of synthesis pathways according to the prior art, formed in the cell by feedback-controlled formation, and the genes coding for the same, are shown in Table 1. In addition, the EC numbers are indicated, which characterize the respective reaction with respect to the substrates and products of the mechanism of the key reaction. In addition, the accession numbers for sequences of the regulated feedback-resistant enzymes of the wild type of E. coli and C. glutamicum are indicated. The nucleotide sequences of these genes and the coded polypeptide sequences are stored in public databases. For C. glutamicum, for example, in the National Center for Biotechnology Information (NCBI) database of the National Library of Medicine (Bethesda, Md., USA) under accession numbers NC_003450.2 and BX927148.1 to BX927157.1. The nucleotide sequences of these genes and the coded polypeptide sequences of E. coli have been described by Blattner et al. (Science 277: 1453-1462 (1997)) and stored in the National Center for Biotechnology Information (NCBI) database of the National Library of Medicine (Bethesda, Md., USA) under accession number NC_000913.2. Access is also possible via the database UniProtKB/Swiss-Prot European Molecular Biology Laboratory, Heidelberg.
TABLE 1Examples of feedback-inhibited enzymes, the enzyme classification numberscharacterizing the same, the genes coding for the same, and the exemplaryaccession numbers of the genes for E. coli and C. glutamicumAccession numberAccession numberEnzymeEC NumberGeneE. coliC. glutamicum2-isopropylmalate synthaseEC 2.3.3.13leuAEG11226YP_224548Acetohydoxy acid synthaseEC 2.2.1.6ilvNEG10502YP_225560.1Acetohydoxy acid synthaseEC 2.2.1.6ilvBEG10494YP_225561.1Acetohydoxy acid synthaseEC 2.2.1.6ilvIEG10500YP_225560.1Acetohydoxy acid synthaseEC 2.2.1.6ilvHEG10499YP_225561.1Acetylglutamate synthaseEC 2.3.1.1argAEG10063YP_225682.1Anthranilate synthaseEC 4.1.3.27trpDEG11027YP_227281.1Anthranilate synthaseEC 4.1.3.27trpEEG11028YP_227280.1Asparagine synthetase BEC 6.3.5.4asnBNP_415200.1NC_006958.1Aspartate transcarbamylaseEC 2.1.3.2pyrBNP_418666Q8NQ38Aspartate transcarbamylaseEC 2.1.3.2pyrINP_418665.1Q8NQ38Aspartate kinaseEC 2.7.2.4lysCEG10550P26512.2Aspartate kinaseEC 2.7.2.4metLEG10590P26512.2Aspartate kinaseEC 2.7.2.4thrAEG10998P26512.2ATP-phosphoribosylEC 2.4.2.17hisGEG10449Q9Z472.2transferaseCarbamoyl phosphateEC 6.3.5.5carAEG10134Q8NSR1.1synthetaseCarbamoyl phosphateEC 6.3.5.5carBNP_414574.1P58939.1synthetaseChorismate mutase IEC 5.4.99.5tyrAEG11039BAB98246.1Chorismate mutase 11EC 5.4.99.5pheAEG10707YP_227138.1Cysteine synthaseEC 2.5.1.47cysKNP_416909.1CBV01938.1Cysteine synthaseEC 2.5.1.47cysMNP_416916.1CBF99279.1D-3-phosphoglycerateEC 1.1.1.95serANP_417388.1AEA48241.1dehydrogenase3-deoxy-D-arabino-EC 4.1.2.15aroGNP_415275.1CAC25920.1heptulosonate-7-phosphatesynthaseDihydrodipicolinate synthaseEC 4.2.1.52dapANP_416973.1CAA37940.1Glutamate dehydrogenaseEC 1.4.1.4gdhNP_416275.1NP_601279.1Glutamate synthaseEC 1.4.1.13gltBNP_417679.2YP_224481.1Glutamate synthaseEC 1.4.1.13gltDNP_417680.1YP_226992.1Glutamine synthetaseEC 6.3.1.2glnANP_418306.1YP_226471.1Glutamyl kinaseEC 2.7.2.11proBNP_414777.1YP_226601.1Homocysteine transmethylaseEC 2.1.1.14metENP_418273.1CAF19845.1Homoserine O-EC 2.3.1.46metXNP_417417.1NP_600817.1aceyltransferaseHomoserine O-EC 2.3.1.46metANP_418437.1YP_225473.1succinyltransferaseHomoserine dehydrogenaseEC 1.1.1.3metLEG10590CAF19887.1Homoserine dehydrogenaseEC 1.1.1.3thrAEG10998NP_414543.1Methionine synthaseEC 2.1.1.13metHNP_418443.1NP_600723.1PhosphoribosylpyrophosphateEC 2.7.6.1prsNP_415725.1YP_225235.1synthetasePrephenate dehydrogenase IEC 1.3.1.12pheANP_417090.1CAF20922.1Prephenate dehydrogenase IIEC 1.3.1.12tyrAEG11039YP_227138.1Pyrroline-5-carboxylateEC 1.5.1.2proCNP_414920.1NP_599858.1reductaseRibose 1,5-bisphosphokinaseEC 2.7.4.23phnNNP_418518.1NP_600170Serine acetyl(succinyl)EC 2.3.1.30cysENP_418064.1NP_601761.1transferaseThreonine ammonia-lyaseEC 4.3.1.19ilvANP_418220.1NP_601328.2Tyrosine aminotransferaseEC 2.6.1.57tyrBNP_418478.1NP_599471.2
Proceeding from the wild form of the feedback-regulated enzymes mentioned in Table 1 by way of example, there is a need to achieve deregulation of the enzyme activity so as to improve the production of target compounds, such as amino acids, nucleotides, amino acid derivatives or intermediates of the synthesis pathways.
It is known that such feedback-resistant enzymes, the regulation of which has been eliminated so as to contribute to improving the performance capability of microorganisms, is important, and feedback-resistant alleles carrying mutations as compared to the wild type have been described. For example, it is described in DE102008040352A1 and EP000002147972A1 that alleles of aroG coding for 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase led to improved formation of tryptophan. Moreover, a recombinant, L-lysine-producing bacterium is known, for example, containing an lysC allele with feedback-resistant aspartate kinase (EP0381527A1). In addition, a recombinant, L-phenylalanine-producing bacterium with feedback-resistant prephenate dehydrogenase is described (EP088424A2). Moreover, a leuA allele is described, which codes for a mutated isopropylmalate synthase and a microorganism producing L-leucine (EP000001568776B1). A mutant acetolactate synthase having a mutation in the IlvN subunit and a microorganism overproducing L-valine are described in EP07017918.9. An allele is also described for the enzyme acetylglutamate synthase, which is feedback-resistant to arginine as compared to the wild type enzyme, and strains that produce more L-arginine with the allele are described (U.S. Pat. No. 7,169,586 B2). Mutant alleles of phosphoribosyl pyrophosphate synthetase and methods for producing L-histidine are described (EP 1529839 A1). It is also known that L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are inhibited by L-histidine, and therefore the ability to produce L-histidine can also be effectively enhanced by introducing a mutation conferring resistance to feedback inhibition into ATP phosphoribosyl transferase (Russian patents nos. 2003677 and 2119536). Improved arginine production was achieved when the enzyme used was carbamoyl phosphate synthetase (EP000001026247A). The introduction of a cysE allele coding for a serine O-acetyltransferase having reduced feedback inhibition through L-cysteine also increased the production of cysteine (US6218168B1; Nakamori et al., 1998, Appl. Env. Microbiol. 64: 1607-1611; Takagi et al., 1999, FEBS Lett. 452: 323-327). A feedback-resistant CysE enzyme substantially decouples the production of O-acetyl-L-serine, the immediate precursor of L-cysteine, from the L-cysteine level of the cell.
Mutagenesis, selection, and mutant selection methods are employed to eliminate the control mechanisms and improve the performance characteristics of these microorganisms. In this way strains are obtained, which are resistant to antimetabolites, such as alpha-amino-beta-hydroxyvaleric acid (AHV), an analog of threonine, or are auxotrophic for regulatorily significant metabolites, and produce amino acids, such as L-threonine. Resistance to 5-methyl-DL-tryptophan (5-MT), a tryptophan analog, is characteristic of a strain producing L-tryptophan, for example (DE 102008040352 A1). In addition, strains that were resistant to 4-aza-D,L-leucine or 3-hydroxy-D,L-leucine, β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine and 5,5,5-trifluoroleucine included a feedback-resistant isopropylmalate dehydrogenase (EP 1568776A2, EP 1067191 A2). Strains are described having acetolactate synthases which exhibit reduced feedback inhibition through the use of the inhibitors sulfonylurea and imidazolinone (CA 2663711 A1). It has also been possible to obtain strains with argA alleles that are suitable for producing L-arginine by selecting slowly growing mutants (U.S. Pat. No. 7,169,586 B2). Likewise, arginine producers were obtained via the resistance-conferring substances 5-azauracil, 6-azauracil, 2-thiouracil, 5-fluorouracil, 5-bromouracil, 5-azacytosine, 6-azacytosine, arginine hydroxamate, 2-thiouracil and 6-azauracil (Japanese patent 49-126819). By utilizing analogs of pyrimidine, such as azidothymidine or azidodeoxyurldine, it is possible to obtain strains in which feedback-resistant enzymes, such as the aspartate transcarbamoylase of the shared synthesis pathway of L-histidine, purine and pyrimidine are mutated (U.S. Pat. No. 5,213,972). By utilizing analogs of lysine, such as S-(2-aminoethyl)-cysteine, it has been possible to obtain lysC alleles, or by utilizing analogs of methionine, such as alpha-methylmethionine, ethionine, norleucine, N-acetylnorleucine, S-trifluoromethylhomocysteine, 2-amino-5-heprenoit acid, selenomethionine, methionine sulfoximine, methoxine, 1-aminocyclopentane carboxylic acid, it has been possible to obtain strains with alleles that accumulate L-methionine at an increased level (EP 1745138 B1). Through the use of 1,2,4-triazole-D-alanine, it has been possible to eliminate the feedback inhibition of the HisG allele hisG13 and thereby obtain strains having enhanced performance characteristics (Mizukami T et al., Biosci Biotechnol Biochem. 1994 April, 58(4):635-8).
A major drawback in the existing production of enzymes having reduced feedback resistance is the use of analogs, such as alpha-amino-beta-hydroxyvaleric acid, 5-methyl-DL-tryptophan, 4-aza-D,L-leucine or 3-hydroxy-D,L-leucine, β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine, sulfonylurea, imidazolinone, 5-azauracil, 6-azauracil, 2-thiouracil, 5-fluorouracil, 5-bromouracil, 5-azacytosine, 6-azacytosine, arginine hydroxamate, 2-thiouracil, 6-azauracil, azidothymidine, azidodeoxyuridine, S-(2-aminoethyl)-cysteine, alpha-methyl-methionine, ethionine, norleucine, N-acetylnorleucine, S-trifluoromethylhomocysteine, 2-amino-5-heprenoit acid, selenomethionine, methionine sulfoximine, methoxine, 1-aminocyclopentane carboxylic acid, 1,2,4,-triazole-D-alanine and the isolation of mutants resistant to such analogs. Another major drawback of existing methods for producing feedback-resistant enzymes, and of the use thereof to enhance the performance capability of the microorganisms, is that many strains must be tested for enhanced performance capability following undirected mutagenesis and the use of analogs, since resistance to analogs can have a wide variety of causes, such as improved decomposition of the analog, or improved export of the analog, which can feign a feedback resistance and results in no new feedback-resistant enzymes being produced, and no strain having enhanced performance capability being present. Existing techniques for isolating the targeted production of feedback-resistant enzymes, which are directed to enhancing the performance capability of microorganisms for the production of amino acids, can therefore achieve the object only partially, incompletely or not at all.